Discovery of 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999): A Highly Selective Mechanism-Based Myeloperoxidase Inhibitor for the Treatment of Cardiovascular Diseases

Article abstract of DOI:10.1021/acs.jmedchem.5b00963

Myeloperoxidase (MPO) is a heme peroxidase that catalyzes the production of hypochlorous acid. Clinical evidence suggests a causal role for MPO in various autoimmune and inflammatory disorders including vasculitis and cardiovascular and Parkinson's diseases, implying that MPO inhibitors may represent a therapeutic treatment option. Herein, we present the design, synthesis, and preclinical evaluation of N1-substituted-6-arylthiouracils as potent and selective inhibitors of MPO. Inhibition proceeded in a time-dependent manner by a covalent, irreversible mechanism, which was dependent upon MPO catalysis, consistent with mechanism-based inactivation. N1-Substituted-6-arylthiouracils exhibited low partition ratios and high selectivity for MPO over thyroid peroxidase and cytochrome P450 isoforms. N1-Substituted-6-arylthiouracils also demonstrated inhibition of MPO activity in lipopolysaccharide-stimulated human whole blood. Robust inhibition of plasma MPO activity was demonstrated with the lead compound 2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999, 8) upon oral administration to lipopolysaccharide-treated cynomolgus monkeys. On the basis of its pharmacological and pharmacokinetic profile, PF-06282999 has been advanced to first-in-human pharmacokinetic and safety studies.

Full text of DOI:10.1021/acs.jmedchem.5b00963

Article
pubs.acs.org/jmc
Discovery of 2‑(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-
dihydropyrimidin-1(2H)‑yl)acetamide (PF-06282999): A Highly
Selective Mechanism-Based Myeloperoxidase Inhibitor for the
Treatment of Cardiovascular Diseases
Roger B. Ruggeri,* Leonard Buckbinder, Scott W. Bagley, Philip A. Carpino, Edward L. Conn,
Matthew S. Dowling, Dilinie P. Fernando, Wenhua Jiao, Daniel W. Kung, Suvi T. M. Orr, Yingmei Qi,
Benjamin N. Rocke, Aaron Smith, Joseph S. Warmus, Yan Zhang, Daniel Bowles, Daniel W. Widlicka,
Heather Eng, Tim Ryder, Raman Sharma, Angela Wolford, Carlin Okerberg, Karen Walters,
Tristan S. Maurer, Yanwei Zhang, Paul D. Bonin, Samantha N. Spath, Gang Xing, David Hepworth,
Kay Ahn, and Amit S. Kalgutkar
Worldwide Research and Development, Pfizer, Inc., Groton, Connecticut 06340, United States
ABSTRACT: Myeloperoxidase (MPO) is a heme peroxidase that
catalyzes the production of hypochlorous acid. Clinical evidence suggests
a causal role for MPO in various autoimmune and inflammatory
disorders including vasculitis and cardiovascular and Parkinson’s diseases,
implying that MPO inhibitors may represent a therapeutic treatment
option. Herein, we present the design, synthesis, and preclinical
evaluation of N1-substituted-6-arylthiouracils as potent and selective
inhibitors of MPO. Inhibition proceeded in a time-dependent manner by
a covalent, irreversible mechanism, which was dependent upon MPO
catalysis, consistent with mechanism-based inactivation. N1-Substituted-
6-arylthiouracils exhibited low partition ratios and high selectivity for
MPO over thyroid peroxidase and cytochrome P450 isoforms. N1-
Substituted-6-arylthiouracils also demonstrated inhibition of MPO
activity in lipopolysaccharide-stimulated human whole blood. Robust inhibition of plasma MPO activity was demonstrated
with the lead compound 2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-
06282999, 8) upon oral administration to lipopolysaccharide-treated cynomolgus monkeys. On the basis of its pharmacological
and pharmacokinetic profile, PF-06282999 has been advanced to first-in-human pharmacokinetic and safety studies.
elevated plasma MPO levels are associated with risk of major
adverse cardiovascular events in otherwise apparently healthy
individuals. In addition, elevated MPO levels are also associated
with risk of secondary adverse events in stable coronary artery
disease subjects, as well as those with recent acute coronary
syndrome.9,10 Mechanisms by which MPO may contribute to
the pathogenesis of diseases include (1) MPO-specific
oxidation of lipids and proteins including those in low-density
and high-density lipoproteins, contributing to oxidative tissue
damage and atherosclerosis;11,12 (2) consumption of nitric
oxide, thereby impairing nitric oxide-dependent vasodilation,
leading to endothelial/vasomotor dysfunction in humans;13,14
and (3) microvascular occlusion resulting from the MPO-
dependent formation of neutrophil extracellular traps in
response to certain stimuli (e.g., phorbol myristate acetate).15
Studies in MPO deficient mice have demonstrated protection
from cardiac remodeling and impaired left ventricular function
INTRODUCTION
■
Myeloperoxidase (MPO, EC 1.11.2.2) is a member of the heme
peroxidase family of enzymes and catalyzes the production of
hypochlorous acid (HOCl) from hydrogen peroxide (H2O2)
and chloride ion (Cl−). MPO is produced in the bone marrow
and stored in the azurophilic granules of neutrophils, where it
constitutes up to 5% of the cellular protein; it is also found,
albeit to a lesser extent, in some human monocytes and
macrophages.1 Upon phagocyte activation, MPO activity
appears within the phagolysosome and extracellularly. The
main function of MPO is considered to be microbicidal;
however, MPO deficiency occurs in ∼1/2000 individuals2 and
is not usually associated with increased risk of infections.3
Compelling human data implicate MPO in the pathogenesis
of a number of acute and chronic inflammatory diseases such as
chronic obstructive pulmonary disease (COPD),4 rheumatoid
arthritis,5 acute kidney injury,6 and Parkinson’s7 and
cardiovascular diseases.8 The role of MPO in cardiovascular
diseases has been particularly well studied in humans; high
levels of MPO are found in atherosclerotic plaques, and
Received: June 22, 2015
© XXXX American Chemical Society
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following surgically induced myocardial infarction.16,17 More
recently, Churg et al.4 have also demonstrated that the MPO
inhibitor 3-[[(2S)-tetrahydrofuran-2-yl]methyl]-2-thioxo-7H-
purin-6-one (AZD-5904,18 AZ1, or TX4, Figure 1) is able to
chlorination (or halogenation) cycle, compound I reacts with
chloride (or other halides) through a two-electron reduction
reaction to be reduced back to the native enzyme, generating
HOCl (or HOX, reaction 2 in Figure 2). Alternatively, in the
peroxidation cycle, compound I may oxidize various electron-
rich organic substrates including phenols,20 anilines,21 and
thiols22 (AH) via a one-electron transfer reaction generating
the compound II form of the enzyme (reaction 3 in Figure 2).
Transfer of an additional electron to compound II from
another molecule of the organic substrate, or an alternative
source, regenerates native MPO (reaction 4 in Figure 2).
While MPO inhibitors hold much promise as therapeutic
agents, their development has been challenging. Many known
MPO inhibitors20,23 such as hydroxamic acid, indole, hydrazine,
aniline, and tryptamine derivatives inhibit the chlorinating
activity of MPO in vitro by acting as peroxidase substrates and
diverting the enzyme from the chlorinating cycle to the
peroxidation cycle. Such compounds promote the formation of
compound II by being readily oxidized by compound I (i.e.,
reaction 3 in Figure 2), one of the strongest oxidizing enzyme
intermediates found in vivo, but they do not then react with
compound II. Accumulated compound II can be easily reduced
back to the native enzyme form by many other electron donors
such as urate, ascorbate, tyrosine, or superoxide anion readily
found in vivo (i.e., reaction 4 in Figure 2).24−28 Thus, in vivo
the mode of inhibition by these inhibitors is reversible and
likely to be ineffective against HOCl-mediated tissue damage.29
More recently, 2-thioxanthines represented by AZD-5904 (also
reported as AZ1 or TX4) and compound A were reported to be
mechanism-based inactivators of MPO (Figure 1).4,30−32 AZD-
5904 and A are suicide substrates of MPO, acting as substrates
in reaction 3 in Figure 2 via the one-electron reduction of
compound I to compound II followed by covalent modification
of the heme moiety by a thiyl radical species generated from
AZD-5904 and A in the course of catalysis.30−32 The resulting
covalent modification of the MPO heme “traps” MPO in an
unreactive state, which cannot readily be converted back to the
native form, in contrast to the reversible single electron transfer
mechanism of inhibition.
Figure 1. Structures of previously reported MPO irreversible
inhibitors.
halt the progression of emphysema and small airway
remodeling in a guinea pig model of COPD. In addition, a
further MPO inhibitor (AZD-3241) is reported to be currently
undergoing clinical trials for the treatment of multiple system
atrophy.19
MPO is a complex hemoprotein with multiple intermediate
states (Figure 2). The peroxidation reaction (reaction 1 in
Figure 2) involves the native ferric enzyme, which undergoes a
two-electron oxidation reaction with H2O2 producing the
highly reactive form of the enzyme, compound I. In the
In the present manuscript, we describe the design, synthesis,
and preclinical characterization of N1-substituted-6-aryl-2-
thiouracil analogs as novel mechanism-based inhibitors of
MPO with high selectivity over thyroid peroxidase (TPO) and
over heme-containing cytochrome P450 (CYP) isoforms. The
lead compound 2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thi-
oxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999,
8) displayed excellent oral pharmacokinetics in preclinical
species and robust irreversible inhibition of plasma MPO
activity both in human blood stimulated exogenously and in
plasma collected after oral (po) administration to lip-
opolysaccharide (LPS)-treated cynomolgus monkeys. PF-
06282999 has been advanced into first-in-human pharmacoki-
netics and safety studies.
Figure 2. Enzymatic cycle for myeloperoxidase. Oxidation of native
MPO by hydrogen peroxide (reaction 1) gives rise to compound I, the
most oxidatively reactive state for MPO. In the halogenation cycle
(reaction 2) a substrate, typically chloride, undergoes oxidation to
afford a reactive species (e.g., hypochlorous acid) that can contribute
to tissue damage under inflammatory conditions. In the peroxidation
cycle, compound I reacts with an electron-rich organic substrate
(reaction 3) to provide a radical species and compound II. For certain
substrates such as those described in this article, the resulting radical
species may then react with MPO to form a covalent adduct, rendering
it irreversibly inhibited. Compound II may also react with another
readily oxidized species (reaction 4) to return to the native state of the
enzyme and complete the peroxidation cycle.
RESULTS AND DISCUSSION
■
Selection of in Vitro Assays To Guide Structure−
Activity Relationship (SAR) Studies. Propylthiouracil
(PTU, Figure 1) is an inhibitor of TPO used clinically for
the treatment of hyperthyroidism.33 Reports on the MPO
substrate and inhibitory properties of PTU have been
published;34−37 however, a detailed kinetic investigation
providing evidence for mechanism-based inactivation of MPO
has not been reported. To assess the feasibility of PTU as a
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potential chemical lead for the de novo design of selective
MPO inhibitors, we examined the mechanism of MPO
inhibition by PTU in greater detail by measuring MPO
peroxidase activity with Amplex Red (10-acetyl-3,7-dihydroxy-
phenoxazine) as a substrate.32
As shown in Figure 3A, the MPO reaction with H2O2 and
Amplex Red was linear over ∼500 s in the absence of PTU.
However, in the presence of PTU, the progressive curves
exhibited curvature and time-dependent inhibition, as typically
observed with an irreversible mechanism of inhibition. The data
were fit to eq 1, and the rate of MPO inactivation at each
inhibitor concentration (kobs) was determined as described in
the Experimental Section. Plotting these kobs values as a
function of PTU concentration revealed a linear line that
passed through near the origin, which was consistent with a
two-step inactivation kinetics process when the concentration
of inhibitor is far below KI (Figure 3B). On the basis of this
model (eq 3), the slope of the line (which is equal to kinact/KI,
the second-order rate constant and the overall inactivation
potency) was determined to be 360 M−1 s−1 for PTU. An IC50
value of 2.81 μM for MPO inhibition was also obtained by
using initial rates from the first 300 s of the reaction at each
inhibitor concentration (Figure 3C). To verify that the
inhibition of MPO by PTU was irreversible and required
H2O2, we performed rapid dilution experiments in the presence
and absence of H2O2. After preincubation of MPO with PTU
for 15 min, the reaction mixture was rapidly diluted 300-fold
into the assay buffer containing both H2O2 and Amplex Red
substrates, and the resulting recovery of MPO activity was
immediately monitored. As shown in Figure 4, no recovery of
MPO activity was observed with PTU when H2O2 was included
in the preincubation mixture, confirming that PTU is an
irreversible inhibitor. However, when the rapid dilution
experiment was performed in the absence of H2O2 substrate
during preincubation, MPO activity was recovered to a similar
level as that of the DMSO control, indicating MPO catalysis by
the substrate H2O2 was required for inhibition by PTU. Taken
together, these data indicated that PTU is a mechanism-based
inhibitor of MPO. TPO inhibition by PTU was evaluated using
methods analogous to those above for MPO and the kinact/KI
ratio and IC50 values were determined to be 442 M−1 s−1 and
3.38 μM, respectively, thus demonstrating that PTU is not
selective as it inhibits both TPO and MPO with comparable
potency.
Although PTU was not selective for inhibition between TPO
and MPO, its mechanism-based inhibition profile was deemed
suitable for a chemical starting point to identify selective
inhibitors, and the assays used in its characterization were
adapted for use as primary in vitro screens for inactivation of
MPO and for selectivity versus TPO. SAR studies were carried
out using the second-order rate constant kinact/KI ratio as the
measure of potency. Unlike IC50 values, kinact/KI ratios are
independent of incubation times and substrate concentrations
and are generally considered the best measure of potency for
irreversible inhibitors.38
Partition ratio is a quantitative assessment of the efficiency
with which mechanism-based inhibitors inactivate their targets.
The reactive intermediate that is formed by reaction of an
enzyme with a potential mechanism-based inhibitor may either
(a) directly form a covalent adduct in the enzyme active site
(resulting in inactivation) or (b) escape from the active site
(leaving enzyme activity intact). PTU was found to have a high
partition ratio of 75 for inhibition of MPO, meaning that the
Figure 3. Inhibition of MPO by propylthiouracil. The MPO reactions
were performed as described in Experimental Section. Data are
averages, and error bars represent the SD from two separate
experiments. (A) Progress curves for MPO inhibition by PTU varied
at 0.47−30 μM. The kobs values were obtained by fitting the time
course as described in Experimental Section. (B) The kobs values were
fit to eq 3 to determine the overall potency kinact/KI ratio. (C) The
initial rates from the first linear ∼300 s of each reaction progress curve
in (A) were used to calculate percent inhibition. The data were fit to
equation, y = 100/[1 + (x/IC50)z], where IC50 is the inhibitor
concentration at 50% inhibition and z is the Hill slope.
activated inhibitory species proceeded to form a covalently
inactivated MPO−PTU adduct only one time for every 76
turnover events of the MPO reaction with PTU. To determine
the partition ratio, the percentage of control activity after rapid
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lipopolysaccharide was also assessed.39 The human whole
blood assay provided a time-averaged inhibition of enzyme
activity in the presence of potentially confounding factors (e.g.,
blood-borne electron donors, plasma protein binding, com-
partmental partitioning). While it remains to be established, it
was assumed that the IC50 values obtained in human whole
blood would correlate with the relative efficacious plasma
concentrations in humans. Also uncertain at the outset was how
well the human MPO biochemical assay (kinact/KI ratio) would
predict improvements in partition ratio or the human whole
blood assay.
Chemistry. N1-Substituted-2-thiouracils have been previ-
ously synthesized via the condensation of appropriate mono-N-
substituted-thiourea and β-ketoester derivatives to afford the
corresponding N1- and N3-substituted-thiouracils as a mixture
of regioisomers.40 In the present work, the synthesis of the
targeted N1-substituted-6-aryl-2-thiouracils was achieved by
employing a regioselective two-step route (Scheme 1) involving
the condensation of the appropriate β-ketoester intermediates
(9−12) with the requisite primary amines (13−17) to yield the
corresponding β-aminoacrylates (18−24). The β-ketoesters 11
and 12 were obtained via the reaction of ethyl hydrogen
malonate (29) and the appropriate benzoic acid derivatives
(intermediates 30 and 31) in the presence of magnesium
ethoxide and 1,1′-carbonyldiimidazole. The corresponding N1-
substituted-thioruracil analogs (2, 3, 5, 7, 8, 25, and 26) were
thus obtained in a regiospecific manner by treatment of the β-
aminoacrylates with isothiocyanatotrimethylsilane.41 Amine-
containing side chain analogs were obtained by postcyclization
modifications. Hydrochloric acid-catalyzed cleavage of the tert-
butyloxycarbonyl protecting group in thiouracil 25 yielded 4 as
the hydrochloride salt. Alternatively, hydrolysis of the thiouracil
ester 26 yielded the carboxylic acid 27, which was then
transformed to 28 by amide coupling with tert-
butyloxycarbonylethylenediamine. Acid-catalyzed cleavage of
the tert-butyloxycarbonyl protecting group in 28 generated
amine 6 as the corresponding hydrochloride salt.
Figure 4. Reversibility of MPO inhibition by PTU. MPO was
▲
●
incubated with 24 μM PTU with ( ) or without ( ) 2 μM H2O2, or
■
◆
DMSO for control with ( ) or without ( ) 2 μM H2O2. After 15
min, an aliquot of the preincubation mixture was rapidly diluted 300-
fold into the MPO reaction buffer containing 2 μM H2O2 and 30 μM
Amplex Red substrates and the MPO activity was immediately
monitored.
dilution was plotted as a function of the ratio of PTU versus
MPO ([PTU]/[MPO]). As shown in Figure 5, the remaining
SAR Studies. Having established assays for the character-
ization of PTU inhibition of MPO and TPO, we utilized this
compound as our starting point to seek thiouracil-based
inhibitors with increased selectivity for MPO and decreased
partition ratio. Initial SAR studies focused on replacement of
the C6-propyl group and on introduction of an N1-substituent.
Concurrently, considerations of the thiourea substructure
contained within the thiouracil (see below) also led to a
focus on a narrowly constrained target physicochemical
properties space (roughly log D < 1.5 to minimize CYP
oxidative metabolism and nonproductive reactive metabolite
formation while keeping PSA < 100 to maintain favorable
membrane permeability and oral absorption) and the
incorporation of specific polar functional groups in the N1-
substituent.
In the course of our present SAR studies (Table 1), it was
observed that replacement of the propyl group in PTU with an
electron-rich aromatic functionality, such as the 2-methox-
yphenyl group in 1, improved kinact/KI ratio by 6-fold for MPO
inhibition, although with only a modest improvement in
inhibitory selectivity for MPO relative to TPO. However, far
greater improvements in selectivity were obtained (>3000-fold)
by substitution of the N1-position on the thiouracil ring, as
shown in 2, which nearly abolished TPO inhibitory activity
(kinact/KI < 3 M−1 s−1, IC50 > 100 μM) while significantly
increasing MPO kinact/KI ratio by 21-fold from PTU.
Figure 5. Determination of the partition ratio for mechanism-based
inactivation of MPO by PTU. MPO was incubated with 2 μM H2O2
and PTU at concentrations varying by 1.5-fold between 0.026 and 12
μM (or DMSO for control). After 15 min, an aliquot of the
preincubation mixture was rapidly diluted 300-fold into the assay
buffer containing 2 μM H2O2 and 30 μM Amplex Red substrates and
the MPO activity was immediately monitored. The initial rate of each
progress curve was determined, and the fractional MPO activity of
each inhibited reaction was plotted as a function of the ratio of PTU to
MPO ([PTU]/[MPO]). Data are averages, and error bars represent
the SD from two separate experiments.
fractional activity decreased as a linear function of [PTU]/
[MPO] until there was no activity remaining. The point at
which this straight line intersected the x-axis was determined to
be 76, which is equal to the partition ratio plus 1. For SAR
purposes, the assay to determine partition ratios provided a
measure of progress in the design of more efficient inhibitors
(lower partition ratio). Higher efficiency inactivation was
expected to be advantageous for in vivo efficacy and for safety
(by minimizing the frequency of reactive intermediates
escaping the MPO active site).
Concurrent with evaluation of compound potency utilizing
biochemical assays, inhibition of MPO in a cell-based assay
employing human whole blood stimulated with bacterial
D
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a
Scheme 1. Synthesis of N1-Substituted-6-arylthiouracil Derivatives Described in This Study
a
Reagents and conditions: (a) Mg ethoxide, CDI; (b) AcOH, 80 °C; (c) TMSNCS; (d) HCl; (e) NaOH; (f) NH2CH2CH2NHBoc, T3P.
While these initial improvements in MPO inhibitory potency
leading from PTU to 1 and 2 were observed employing the
biochemical assay, similar improvements were not seen in the
human whole blood assay (Table 1). Ongoing optimization of
the N1- and C6-substituents showed that both made significant
contributions to potency, and the combination ultimately led to
compounds with improved whole blood potency. The preferred
C6-ring system was found to be disubstituted phenyl, with the
second substituent (methoxy or chloro) at the para (e.g., 2-6)
or meta (e.g., 7 and 8) positions, providing a significant
increase in potency; we concluded that MPO inhibitory
potency was more dependent on the electron-density and
substitution pattern on the C6-phenyl ring than on its overall
hydrophobicity. Significant reductions in whole blood IC50
were not afforded until kinact/KI values were increased to
>10 000 M−1 s−1, although improvements in kinact/KI values did
not then strictly correlate with lower IC50 values in the human
whole blood assay (e.g., alcohol 3 vs amine 4). Factors
diminishing the direct correlation of potencies between the
biochemical and whole blood assays may include differences in
the unbound plasma concentrations (Table 2), distribution
between compartments within whole blood, and/or varying
contributions of the nonproductive single electron transfer
(compound II) pathway in the biochemical assay. As such,
potency improvement in the whole blood assay below 0.5 μM
proved to be highly challenging, especially while operating
within a constrained physiochemical property space (see below
for additional discussion). This may be due to modest affinity
of the inhibitor for the active site (as reflected by the >100 nM
KI values from the Amplex Red assay), competition with high
concentration of native endogenous substrates (e.g., chloride
and H2O2), dynamics of MPO activation in the assay, and/or
efficiency of capture by the activated inhibitor species (partition
ratio).
Concurrent with the development of SAR around sub-
stituents on the thiouracil ring, we initiated planning and
experiments to better understand the risks associated with the
thiouracil functionality. The thiouracil motif is considered a
“structural alert” (analogous to the thiourea functionality)
because of its propensity toward bioactivation to reactive
species.42 For example, cases of immune-mediated hepatotox-
icity and agranulocytosis have been reported in patients
receiving chronic PTU treatment.43−45 There is causal evidence
linking the immune-mediated toxicity to the oxidative
bioactivation of PTU by MPO.34−37 In activated human
polymorphonuclear leukocytes and/or human MPO/H2O2/
Cl−, PTU undergoes oxidative metabolism on its thiocarbonyl
−
motif (Scheme 2) to yield PTU-disulfide, PTU-SO2 , and the
−
protein- and thiol-reactive PTU-SO3 as metabolites. It has
been proposed that modification of neutrophil proteins,
including MPO, may induce the formation of the antineutrophil
antibodies that have been detected in patients with PTU-
induced agranulocytosis46−48 and lupus-like syndrome.49 In our
present studies, liquid chromatography−tandem mass spec-
trometry (LC−MS/MS) analysis of an incubation of
arylthiouracil 2 (exact mass (MH+) of 351.1367) with human
MPO/H2O2/Cl− in the presence of excess glutathione (GSH)
(1 mM) indicated the formation of a GSH conjugate (MH+ =
624.2334), which can be obtained via oxidation of the
thiocarbonyl group in 2 to the putative sulfonate species
E
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Table 1. Representative Set of in Vitro Pharmacology Data for N1-Substituted-6-arylthiouracil Analogs
a
b
log D was measured at pH 7.4 using the previously described shake-flask method.55 kinact and KI values were determined as described in
Experimental Section. Values are the geometric mean [90% CI] of at least two independent experiments. The overall potency (kinact/KI) was
calculated using these mean values. When kinact and KI values could not be determined separately (i.e., KI ≫ [I]), the kinact/KI ratios were obtained
from the slopes, similar to Figure 3B, and values listed are the geometric mean values ( 90% CI) of at least two independent experiments. The kinact
/
KI ratios given for TPO inhibition are geometric mean values [90% CI] that when measurable were obtained by using initial rates from the first 300 s
c
d
of the reaction at each inhibitor concentration. Partition ratios were generated from two to four separate experiments. IC50 values shown are
geometric mean values [90% CI] of individual determinations for inhibition of MPO activity in LPS-stimulated human whole blood.
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Table 2. Representative in Vitro ADME Data for N1-Substituted-6-arylthiouracil Analogs
a
b
c
c
compd PSA (Å2) permeability RRCK Papp (10−6 cm/s)
human PPB fu
HLM CLint,app (μL min−1 mg−1
)
HHEP CLint,app (μL min−1 per 106 cells)
d
d
2
3
4
5
6
7
8
65
76
82
99
111
99
90
26
ND
11
ND
13
0.5
0.7
0.5
0.9
0.6
0.4
<8.0
8.8
<6.0
2.9
3.5
0.8
1.5
1.1
1.6
<8.0
<8.0
<8.0
<8.0
<6.0
<6.0
<6.0
<6.0
a
Passive permeability was determined in Ralph−Russ canine kidney cells, which are a low transporter cell line, isolated from Madine−Darby canine
kidney cells.57 Fraction unbound (fu) in human plasma was determined from equilibrium dialysis method and is a mean value from three individual
b
determinations.58 CLint,app refers to total apparent intrinsic clearance obtained from scaling in vitro half-lives in HLM and HHEP as previously
c
described.63 ND = not determined.
d
Scheme 2. Oxidative Bioactivation of the Thiouracil Motif in
PTU and Compound 2
was not obvious whether such a tactic would compromise the
ability of the compound(s) to inhibit MPO in the desired
irreversible and selective manner. However, optimization of the
N1-substituent demonstrated that small polar groups such as a
hydroxyl (3), amino (4), or amidoyl (5) not only were
tolerated for MPO inhibition but also enhanced the
effectiveness of the arylthiouracil to act as an irreversible
inhibitor, as evidenced by their increased kinact/KI and their
lower IC50 in the human whole blood assay (Table 1). Higher
potencies in the biochemical assay were observed with alcohols
(e.g., 3, kinact/KI = 29 800), while greater inhibitory potencies in
the human whole blood assay were obtained with the amines
(e.g., 4 and 6, human whole blood IC50 of 0.50 and 0.60 μM,
respectively). Although not as robust an MPO potency
improvement when compared with the alcohol and amine
variants, the primary carboxamide functionality (e.g., 5) also
provided another viable alternative for modulating physical
properties in the balance of efficacy and safety. Gratifyingly,
compounds 3−5 also displayed a greatly improved MPO
inactivation efficiency (partition ratio) with 4 possessing the
highest inactivation efficiency (lowest partition ratio) of 4.7
(versus 75 for PTU), which suggested that MPO inactivation
efficiency may be a key determinant of inhibitory activity in the
human whole blood assay.
followed by nucleophilic displacement in the presence of
exogenously added GSH (Scheme 2).
With awareness of the high partition ratio of PTU, we
speculated that systemic exposure to reactive species derived
from oxidative bioactivation of the thiocarbonyl motif (e.g., the
corresponding sulfonate) could be minimized if a pendent
nucleophilic group were tethered to the N1-substituent such
that potential thiocarbonyl reactive species in solution could be
rapidly quenched in an intramolecular fashion. At the outset it
Figure 6. LC−MS/MS analysis of the oxidative metabolism of alcohol 4 to cyclic ether M1 in the presence of H2O2.
G
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Table 3. Preclinical Pharmacokinetics of Selected N1-Substituted-6-arylthiouracil Derivatives
Article
a
b
b
compd
species
CLp (mL min−1 kg−1
)
Vdss (L/kg)
t1/2 (h)
Tmax (h)
1.3 0.6
1.17 0.76
oral F (%)
3
rat
59.3 2.65
0.82 0.02
0.25 0.01
8
dog
rat
11.9 2.87
1.33 0.26
5.3 0.7
16
91
84
76
31
3
4
5
6
7
8
70.3 (79, 69.5)
13.4 (12.3, 14.4)
35.1 (35.8, 34.4)
1.31 (1.44, 1.17)
38.6 5.52
3.71 (4.34, 3.08)
3.66 (3.89, 3.43)
1.23 (1.37, 1.09)
0.30 (0.29, 0.30)
0.59 0.17
1.1 (0.9, 1.2)
5.7 (5.8, 5.7)
0.76 (1.02, 0.50)
3.4 (3.2, 3.6)
0.81 0.02
0.38 (0.25, 0.50)
1.3 (2.0, 0.5)
0.5 (0.5, 0.5)
1.5 (2.0, 1.0)
0.75 (0.5, 1.0)
1.0 (1.0, 1.0)
0.67 0.29
0.63 (1.0, 0.25)
0.78 1.10
1.0 0.1
dog
rat
dog
rat
dog
rat
4.10 (3.81, 4.39)
37.6 1.36
0.86 (0.74, 0.97)
0.83 0.16
2.59 (2.50, 2.68)
0.38 0.19
8
61
100
86
100
75
76
dog
rat
7.07 (7.86, 6.27)
41.8 9.65
1.03 (0.92, 1.14)
2.13 1.08
2.80 (1.70, 3.90)
0.75 0.19
mouse
dog
monkey
10.1 0.83
0.90 0.06
1.36 0.12
3.39 1.13
0.54 0.04
2.2 0.5
0.7 0.3
10.3 (10.5,10.1)
1.09 (1.17, 1.01)
3.28 (3.31, 3.24)
1.5 (1.0, 2.0)
a
All experiments involving animals were conducted in our AAALAC-accredited facilities and were reviewed and approved by Pfizer Institutional
Animal Care and Use Committee. Pharmacokinetic parameters were calculated from plasma concentration−time data and are reported as mean
values ( SD for n = 3 and individual values for n = 2). All pharmacokinetics were conducted in male gender of each species (Wistar rats, CD-1 mice,
beagle dogs, and/or cynomolgus monkey). Intravenous (iv) doses for the test compounds were 1 mg/kg, and compounds were administered in
saline (compounds 4 and 6) or 12% sulfobutyl ether β-cyclodextrin (compounds 3, 4, 5, 7, and 8) solution. Oral (po) doses for the test compounds
b
were either 3 or 5 mg/kg, and compounds were formulated in 0.5% methylcellulose. Obtained from po dose.
Oxidative desulfurization of thioureas to ureas by H2O2 is a
well-studied reaction.50,51 Therefore, an initial assessment of
the viability of the intramolecular trapping of reactive species
with the pendent nucleophile was conducted via reacting
alcohol 3 (10 μM) with H2O2 (500 μM) in phosphate buffer
(pH 7.4) for 60 min at 37 °C. While no sulfonic acid metabolite
could be observed, its presence as an intermediate in the
incubation was inferred from the quantitative conversion of 3
(tR = 17.49 min, MH+ = 309.0903) to the stable cyclic ether M1
(tR = 14.99 min, MH+ = 275.1026) as a metabolite (Figure 6)
upon analysis by LC−MS/MS. The formation of M1 was also
noted in incubations of 3 (10 μM) in the MPO/H2O2/Cl−
system in a H2O2-dependent fashion, although the reaction was
not quantitative likely due to rapid inhibition of MPO activity.
Importantly, no sulfhydryl conjugates of 3 were detected upon
addition of excess GSH (1 mM) to the H2O2 or MPO/H2O2/
Cl− incubations. A similar analysis with thiouracils 4, 6, 7, and 8
in the MPO/H2O2/Cl− system in the presence of excess GSH
also failed to generate GSH adducts. Overall, these observations
support the hypothesis that although an undesired reactive
metabolite may be formed in the course of MPO catalysis, the
intramolecular trapping mechanism provides the potential for
decreasing exposure to the reactive metabolite(s) in vivo.
Besides peroxidases, heme-containing CYPs or other
oxidative enzymes such as flavin monooxygenases in human
hepatic tissues (e.g., human liver microsomes (HLM) and
cryopreserved hepatocytes (HHEP)) are also capable of
oxidizing the thiourea/thiouracil functionalities to reactive
species capable of eliciting a toxicological response.42,52−54 In
the present situation, minimization of oxidative metabolism/
bioactivation of the thiouracil derivatives was largely driven
through a reduction in lipophilicity as determined by
log D7.4.55,56 Aiding in this endeavor was an only modest
dependency between log D and MPO inhibitory potency as
noted earlier. Virtually all compounds prepared with log D <
1.5 were resistant to metabolic turnover in prototypic stability
studies in NADPH-supplemented HLM and HHEP, as
reflected from their very low apparent intrinsic clearance
(CLint,app) values (Table 2). However, while potency and
metabolic clearance tended to favor molecules at the lower
ranges of hydrophobicity, the less-lipophilic analogs obtained
with increasingly polar functionalities (e.g., N-ethylaminoamide
6, PSA = 111) showed impairment in passive membrane
permeability (Papp) as measured in the Ralph−Russ canine
kidney (RRCK) assay57 (Tables 1 and 2). Fortunately, the
range of physicochemical properties discovered to be tolerated
within this series of thiouracils provided sufficient latitude to
allow overall balance to be achieved with regard to absorption,
distribution, metabolism, and excretion (ADME) properties
discussed below.
Pharmacokinetics and in Vitro ADME Profiling. A
subset of N1-substituted-6-arylthiouracils that met the MPO
inhibitory potency and selectivity criteria were advanced to
intravenous (iv) and po pharmacokinetic studies in preclinical
species. The pharmacokinetic parameters describing the
disposition are summarized in Table 3. Following iv
administration to rats, compounds 5−7 demonstrated moder-
ate plasma clearance (CLp) ranging from 35.1 to 38.6 mL
min−1 kg−1, while compounds 3 and 4 exhibited high CLp (59−
70 mL min−1 kg−1), which approached the rat hepatic blood
flow of 70−80 mL min−1 kg−1. In contrast, compounds 3−7
exhibited relatively low CLp (1.31−13.4 mL min−1 kg−1) in
dogs. Furthermore, with the exception of primary alcohol 3 and
basic amine 6, good oral bioavailability (F) was discerned with
4, 5, and 7 in rats and dogs (oral F of 3 and 6 was 8.0−16% in
rats and dogs; oral F of 4, 5, and 7 was 31−100% in rats and
dogs).
In the course of balancing MPO inhibitory potency, TPO
selectivity, and in vivo pharmacokinetics, our focus was drawn
to the series of acetylamides (compounds 6, 7, and 8). At the
lower end of the lipophilicity range, N-ethylaminoamide 6
demonstrated potent inhibition of MPO activity (IC50 = 0.6
μM) in human whole blood. However, clinical viability of 6 was
limited due to its low oral systemic bioavailability in preclinical
species, which may be a consequence of its low membrane
permeability due to its high polarity and PSA (log D7.4 = −1.4,
PSA = 111). Each member of the pair of primary amides
possessing a pendent 2,5-disubstituted phenyl group (com-
H
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Article
pounds 7 and 8) was considered for further investigations due
to their improved MPO inhibitory potency relative to 5, as well
as the observed high oral bioavailability (Table 3). While both
compounds possessed desirable attributes, 8 was advanced into
a preclinical pharmacology study in non-human primates due to
advantages in thermodynamic solubility and predicted human
bioavailability.
antibody coated plates, and following exchange of plasma for
drug-free assay media, the residual activity of the captured
MPO was measured using the peroxidation of Amplex Red. A
mixed effect sigmoid model was applied to study the
relationship between plasma exposure of 8 and the MPO
capture activity at 2 h after dose and 3 h after LPS
administration, which corresponds to the peak of MPO activity.
As shown in Figure 7, the estimated EC50 for total 8
concentration in plasma was 3.8 μM, which corresponds well
with the IC50 value obtained in the human whole blood assay of
1.9 μM.
The in vivo pharmacokinetics of 8 were examined in greater
detail in mice, rats, dogs, and monkeys, wherein it was
demonstrated to have low CLp in mice (10.1 mL min−1 kg−1),
dogs (3.39 mL min−1 kg−1), monkeys (10.3 mL min−1 kg−1)
and moderate CLp in rats (41.8 mL min−1 kg−1). The terminal
plasma elimination half-lives (t1/2) ranged from 0.75 to 3.3 h in
the four species. Approximately 26−32% of the iv dose of 8 was
excreted in the unchanged form in rat, dog, and monkey urine,
wherein it was also shown that it was well distributed with
steady state distribution volumes (Vdss) ranging from 0.5−2.1
L/kg in mice, rats, dogs, and monkeys. Following oral
administration, 8 was rapidly (Tmax = 0.78−1.70 h) and well
absorbed in mice, rats, dogs, and monkeys with oral
bioavailability values of 100%, 86%, 75%, and 76%, respectively.
The mean plasma free fraction (fu) of 8 (2 μM), determined by
equilibrium dialysis,58 in mouse, rat, dog, monkey, and human
was 0.451, 0.447, 0.460, 0.536, and 0.376, respectively. The
ability of 8 to reversibly inhibit the major human CYP enzymes
was investigated in human liver microsomes using established
protocols.59 Likewise, the potential of 8 to inhibit major human
CYP enzymes in a time- and concentration-dependent manner
was assessed using an IC50 shift assay in human liver
microsomes.60 In addition, 8 demonstrated no reversible
inhibition and no change in time-dependent inhibitory potency
(IC50 > 100 μM) against CYP1A2, CYP2B6, CYP2C8,
CYP2C9, CYP2C19, CYP2D6, and CYP3A4 catalytic activities
in human liver microsomes. Consistent with its low lipophilicity
(log D7.4 = 0.81), 8 was resistant toward metabolic turnover in
NADPH-supplemented HLM and cryopreserved HHEP and
was devoid of GSH adduct formation in these biochemical
matrices. On the basis of these observations and physicochem-
ical properties (including low log D and low passive
permeability), we speculate that metabolic elimination will be
inconsequential as a clearance mechanism in humans and that 8
will be principally eliminated via renal excretion in the
unchanged form.61 In addition, 8 was also devoid of mutagenic
responses in the Salmonella Ames and in vitro micronucleus
assays in the absence or presence of CYP activation (aroclor-
induced rat liver S9 fraction/NADPH), and no significant off-
target activity was discerned upon evaluation of 8 (at 100 μM)
across a broad panel of receptors, ion channels, enzymes, and
transporters.
Figure 7. Concentration−effect relationship for inhibition of MPO
activity by 8 in a cynomolgous monkey LPS challenge study.
Compound 8 was administered orally (5, 20, and 80 mg/kg) 1 h
after iv administration of LPS. After 2 h (the peak in MPO activity)
plasma samples were collected and concentrations of 8 as well as
residual MPO activity determined. The plasma concentration of 8 that
correlated with 50% of the maximum MPO inhibition achieved was 3.8
μM, approximately twice the IC50 observed in the human whole blood
assay (1.9 μM).
Summary. We have described the design, synthesis, and
preclinical evaluation of N1-substituted-6-aryl-2-thiouracils as
potent mechanism-based inactivators of MPO with great
improvements in MPO inactivation efficiency (as evidenced
by reduced partition ratio) over PTU. Furthermore, in contrast
with PTU, the N1-substituted-6-aryl-2-thiouracil derivatives
also demonstrated high selectivity over the related haloperox-
idase TPO. Upon the basis of its potent and selective MPO
inhibitory characteristics, lack of oxidative metabolism in HLM
and HHEP, clean off-target profile including lack of measurable
TPO activity, as well as favorable preclinical in vivo safety
results, PF-06282999 (8) has been advanced to clinical trials for
human pharmacokinetics, safety/tolerability, and MPO inhib-
ition studies, which are currently underway and will be reported
in due course.
In Vivo Pharmacology. In order to ascertain whether the
advances noted in the in vitro and ex vivo assays for candidate
thiouracil derivatives translated to effective irreversible
inhibition of MPO in vivo, 8 was also advanced to an in vivo
pharmacology study in cynomolgus monkeys using iv
endotoxin (LPS) challenge, a classic model of inflammatory
leukocyte activation with corresponding MPO activation
demonstrated in various species including human.62 In this
randomized crossover study, cynomolgus monkeys were orally
administered either vehicle or 8 (5, 20, and 80 mg/kg) 1 h after
iv administration of LPS. Blood was sampled throughout the
study and heparinized plasma prepared for MPO activity
measurements as well as determination of 8 plasma
concentrations. Total MPO was captured using anti-MPO
EXPERIMENTAL SECTION
■
MPO Assay and Determination of Inhibitor Potency (kinact/KI
Ratios). MPO was purified from human polynuclear leukocytes, and
MPO peroxidase activity was measured by monitoring the formation
of resorufin generated from the oxidation of Amplex Red by MPO as
described previously.31 Briefly, the assay mixture contained 50 mM
sodium phosphate buffer, pH 7.4, 150 mM NaCl, 1 mM diethylene-
triaminepentacetic acid, 2 μM H2O2, 30 μM Amplex Red, and the
indicated concentrations of inhibitor (or DMSO). Reactions were
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Article
initiated by the addition of 100 pM MPO, and the final concentration
of DMSO was kept at 2%. The first ∼600 s of the reaction progress
curves corresponding to the linear range of the DMSO control were fit
to eq 1.
NMR spectra were recorded on 400 or 500 MHz spectrometers and
are reported relative to residual undeuterated solvent signals. Data for
1H NMR spectra are reported as follows: chemical shift (δ),
multiplicity, coupling constant (Hz), and integration. The peak shapes
are denoted as follows: s, singlet; d, doublet; dd, doublet of doublets; t,
triplet; q, quartet; spt, septet; m, multiplet; br s, broad singlet. Mass
spectrometry (MS) was performed via atmospheric pressure chemical
ionization (APCI) or electron scatter (ES) ionization sources. High-
resolution mass spectrometry (HRMS) was performed via electrospray
ionization (ESI) source. The system used was an Agilent 1200 DAD
(G1315C), 190−400 nm scan, 4 nm slit, and Agilent 6220 MS (TOF).
Where the intensity of single chlorine ions is described, the expected
intensity ratio was observed (approximately 3:1 for 35Cl/37Cl-
containing ions) and the intensity of only the lower mass ion is
given. Elemental analyses were performed by Intertek Pharmaceutical
Services, Whitehouse, NJ. HPLC purity was determined using a
Kinetex C18 column (100 mm × 3.0 mm, 2.6 μm), eluting with 95:5
water/acetonitrile (both solvents containing 0.1% formic acid), flow
rate = 0.75 mL/min, detecting at 215 nm. HPLC purity is reported as
>95% if no peaks other than the desired product were observed.
Ethyl (Z)-3-(2,4-Dimethoxyphenyl)-3-((2-isopropoxyethyl)-
amino)acrylate (18). A solution of ethyl 3-(2,4-dimethoxyphenyl)-
3-oxopropanoate (9) (0.95 g, 4.0 mmol) and 2-isopropoxyethan-1-
amine (13) (1.3 g, 12.0 mmol) in ethanol (4 mL) was treated with
acetic acid (0.70 mL, 12.0 mmol) and then heated at reflux. After 64 h,
the reaction mixture was allowed to cool to ambient temperature and
concentrated under reduced pressure. The resulting residue was
dissolved in dichloromethane (400 mL) and washed sequentially with
an aqueous 1 M HCl solution (100 mL), a saturated aqueous sodium
bicarbonate solution (100 mL), and brine (100 mL). The organic layer
was dried over MgSO4 and concentrated. The resulting crude product
was purified by flash column chromatography on silica gel using a
gradient of 5−10% ethyl acetate in heptane to afford the title
compound 18 (0.75 g, 56%). 1H NMR (400 MHz, CDCl3) δ 8.74 (t, J
= 5.7 Hz, 1 H), 7.12 (d, J = 8.2 Hz, 1 H), 6.48 (dd, J = 8.2, 2.1 Hz, 1
H), 6.45 (d, J = 2.3 Hz, 1 H), 4.46 (s, 1 H), 4.13 (q, J = 7.0 Hz, 2 H),
3.82 (s, 3 H), 3.80 (s, 3 H), 3.55 (spt, J = 6.1 Hz, 1 H), 3.40 (t, J = 6.1
Hz, 2 H), 3.11 (m, 2 H), 1.26 (t, J = 7.1 Hz, 3 H), 1.14 (d, J = 6.3 Hz,
6 H). LCMS (ESI+) m/z: 338.2 [M + H]+ (100%).
6-(2,4-Dimethoxyphenyl)-1-(2-isopropoxyethyl)-2-thioxo-
2,3-dihydropyrimidin-4(1H)-one (2). A mixture of ethyl (Z)-3-
(2,4-dimethoxyphenyl)-3-((2-isopropoxyethyl)amino)acrylate (18)
(0.64 g, 1.9 mmol) and isothiocyanatotrimethylsilane (1.1 mL, 9.4
mmol) was heated at 110 °C by microwave irradiation for 1 h. After
cooling to ambient temperature, the reaction mixture was directly
purified by flash column chromatography on silica gel, eluting with a
5−10% gradient of ethyl acetate in heptane to afford 2 (0.54 g, 80%)
as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 9.88 (br s, 1 H),
7.14 (d, J = 8.4 Hz, 1 H), 6.54 (dd, J = 8.4, 2.3 Hz, 1 H), 6.49 (d, J =
2.1 Hz, 1 H), 5.79 (s, 1 H), 4.63−4.71 (m, 1 H), 3.85 (s, 3 H), 3.81 (s,
3 H), 3.67−3.84 (m, 2 H), 3.48 (ddd, J = 9.8, 5.9, 3.1 Hz, 1 H), 3.45
(spt, J = 6.1 Hz, 1 H), 1.030 (d, J = 6.1 Hz, 3 H), 1.027 (d, J = 6.1 Hz,
3 H). LCMS (ESI+) m/z: 351.2 [M + H]+ (100%). HRMS: m/z calcd
for C17H23N2O4S [M + H]+ 351.1379, found 351.1373. Anal. Calcd for
C17H23N2O4S: C, 58.27; H, 6.33; N, 7.99; S, 9.15. Found: C, 57.81; H,
6.25; N, 7.81; S, 9.18. HPLC purity: >95%.
V
kobs
0
product =
[1 − exp(−kobst)]
(1)
where V0 is the initial rate in RFU/s and t is time in seconds, to obtain
the first order rate constant for enzyme inactivation (kobs) at each
inhibitor concentration. Each kobs value was corrected for auto-
inactivation of the enzyme by subtracting the kobs value for the
uninhibited reaction. The corrected kobs values were then plotted
versus inhibitor concentration ([I]) and fit to eq 2.
k
inact[I]
kobs
=
KI + [I]
(2)
where kinact is the maximal rate of inactivation and KI is the inhibitor
concentration that yields half the rate of maximal inactivation. When
[I] ≪ KI, eq 2 is simplified to eq 3,
k
kobs
=
inact [I]
KI
(3)
where the kinact/KI is calculated from the slope, which is obtained from
the kobs vs [I] linear lines.38 All assays were performed in 96-well,
black, half-area, nonbinding surface, polystyrene plates (Corning,
Tewksbury, MA). Fluorescent changes (relative fluorescent units/s,
RFU/s) were monitored at room temperature every 20 s on a
Spectramax M2 microplate spectrophotometer (Molecular Devices,
Palo Alto, CA) equipped with Softmax Pro software (Molecular
Devices, Palo Alto, CA) with excitation and emission filters set at 530
and 580 nm, respectively. All data were analyzed using nonlinear
regression analysis in Microsoft Excel and Kaleidagraph (Synergy
Software, Reading, PA).
TPO Assay. TPO activity was measured by monitoring the
formation of resorufin from the oxidation of Amplex Red using
conditions similar to those in the described in the MPO assay.39 Assay
mixtures (100 μL) contained 50 mM sodium phosphate buffer, pH
7.4, 150 mM NaCl, 2 μM H2O2, 30 μM Amplex Red, 1 mM
diethylenetriaminepentaacetic acid, and 2% DMSO. The reactions
were initiated by the addition of TPO. Reaction mixtures to determine
the background reaction rate consisted of all assay components and 4
μL of 500 unit/mL bovine catalase in 50 mM potassium phosphate
buffer, pH 7.0. The background rate was subtracted from each reaction
progress curve. All data were analyzed using nonlinear regression
analysis in Microsoft Excel and Kaleidagraph (version 3.5, Synergy
Software).
Human Whole Blood Assay for Irreversible Inhibition of
MPO. MPO activity was determined using modifications to a
described method,62 using human whole blood from healthy
volunteers, collected in heparinized tubes. Test compound was
incubated with human whole blood stimulated with bacterial LPS
for 4 h, followed by capture of MPO on immobilized anti-MPO
antibody coated plates. The captured MPO was washed and residual
MPO activity was determined using Amplex Red and H2O2 as
described in our previous publication.39
Compound Synthesis. All chemicals, reagents, and solvents were
purchased from commercial sources and were used without further
purification. Except where otherwise noted, all reactions were run
under an inert atmosphere of nitrogen gas using anhydrous solvents.
Also, except where otherwise noted, all reactions were run at room
temperature (∼23 °C). The term “concentrated” refers to the removal
of solvent at reduced pressure on a rotary evaporator with a water bath
temperature not exceeding 60 °C. Silica gel chromatography was
performed using a medium pressure Biotage or ISCO system and
columns prepackaged by various commercial vendors including
Biotage and ISCO. PTU and 6-(2-methoxyphenyl)-2-thioxo-2,3-
dihydropyrimidin-4(1H)-one (1) were purchased from commercial
sources. Compound 7 (also known as PF-1355 or PF-06281355) is
commercially available from Sigma-Aldrich (catalog no. PZ0277).
Methyl (Z)-3-(2,4-Dimethoxyphenyl)-3-((2-hydroxyethyl)-
amino)acrylate (19). A mixture of methyl 3-(2,4-dimethoxyphen-
yl)-3-oxopropanoate (10) (3.50 g, 14.69 mmol) and acetic acid (0.17
mL, 2.94 mmol) in isopropanol (70 mL) was combined with
ethanolamine (0.88 mL, 14.69 mmol) and heated to 80 °C. After 2, 4,
and 6 h, additional ethanolamine (0.88 mL, 14.69 mmol) was added to
the reaction mixture. After 48 h, the reaction mixture was cooled to
ambient temperature and then concentrated under reduced pressure.
The resulting residue was suspended in equal parts of a saturated
sodium bicarbonate solution and water under N2. After stirring
overnight, the solids were filtered and dried in a vacuum oven at 30 °C
1
overnight to afford 19 (2.72 g, 63%) as a beige colored power. H
NMR (400 MHz, CDCl3) δ 8.77 (t, J = 5.4 Hz, 1 H), 7.13 (d, J = 8.3
Hz, 1 H), 6.47−6.52 (m, 2 H), 4.53 (s, 1 H), 3.84 (s, 3 H), 3.82 (s, 3
J
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Article
H), 3.66 (s, 3H), 3.61 (td, J = 5.5, 5.5 Hz, 2 H), 3.15 (td, J = 5.5, 5.5
Hz, 2 H).
= 2.2 Hz, 1 H), 5.81 (d, J = 2.2 Hz, 1 H), 4.68−4.81 (m, 2 H), 3.87 (s,
3 H), 3.84 (s, 3 H), 3.74 (dt, J = 14.4, 5.4 Hz, 1 H), 3.23−3.45 (m, 2
H), 1.40 (s, 9 H). LCMS (ESI+) m/z: 408.3 [M + H]+ (100%).
1-(2-Aminoethyl)-6-(2,4-dimethoxyphenyl)-2-thioxo-2,3-di-
hydropyrimidin-4(1H)-one Hydrochloride (4). Acetyl chloride
(55 mL, 770 mmol) was added slowly over 3 min to a solution of
ethanol (50 mL, 860 mmol) in ethyl acetate (390 mL), cooled by
stirring in an ice/water bath. After 5 min, the cooling bath was
removed, and the solution was stirred for an additional 45 min before
being added to tert-butyl 2-(6-(2,4-dimethoxyphenyl)-4-oxo-2-thioxo-
3,4-dihydropyrimidin-1(2H)-yl)ethylcarbamate (25) (31.7 g, 77.8
mmol). After stirring for 5 h, the resulting solid was collected by
vacuum filtration, rinsing with ethyl acetate. The material collected was
dried under vacuum to afford 26.6 g (99.3%) of the desired product 4
6-(2,4-Dimethoxyphenyl)-1-(2-hydroxyethyl)-2-thioxo-2,3-
dihydropyrimidin-4(1H)-one (3). A solution of methyl (Z)-3-(2,4-
dimethoxyphenyl)-3-((2-hydroxyethyl)amino)acrylate (19) (9.50 g,
33.8 mmol) in 2-methyltetrahydrofuran (100 mL) was treated with
isothiocyanatotrimethylsilane (23.80 mL, 168.79 mmol), and the
resulting reaction mixture was heated at 85 °C. After stirring overnight,
the reaction mixture was cooled to ambient temperature, extracted
with an aqueous 1 N NaOH solution (1 × 250 mL, then 1 × 50 mL);
the combined aqueous layers were washed with methylene chloride (2
× 50 mL), and the aqueous phase was acidified to pH 4 with
concentrated HCl. The resulting solids were collected by filtration,
washed with water (2 × 50 mL), and dried under N2 overnight to
afford a light yellow powder. This material was then dissolved in DMF
(70 mL) at 90 °C before water (80 mL) was added to the hot solution.
After allowing this solution to cool to ambient temperature and stirring
overnight, the resulting solids were filtered, washed with water, and
dried under high vacuum to provide 3 (6.7 g, 61%) as an off-white
powder. 1H NMR (500 MHz, DMSO-d6) δ 12.68 (s, 1 H), 7.24 (d, J =
8.3 Hz, 1 H), 6.69 (d, J = 2.4 Hz, 1 H), 6.65 (dd, J = 8.4, 2.3 Hz, 1 H),
5.70 (d, J = 2.2 Hz, 1 H), 4.69 (t, J = 4.9 Hz, 1 H), 4.50 (ddd, J = 13.4,
7.1, 4.2 Hz, 1 H), 3.83 (s, 3 H), 3.82 (s, 3 H), 3.59 (dt, J = 13.4, 7.3
Hz, 1 H), 3.46−3.55 (m, 1 H), 3.38−3.46 (m, 1 H). MS (ESI+) m/z:
309.1 [M + H]+. HRMS: m/z calcd for C14H17N2O4S [M + H]+
309.0909, found 309.0906. Anal. Calcd for C14H16N2O4S: C, 54.53; H,
5.23; N, 9.09; S, 10.40. Found: C, 54.29; H, 5.14; N, 8.98; S, 10.43.
HPLC purity: 98.1%.
Ethyl (Z)-3-(2-(tert-Butoxycarbonylamino)ethylamino)-3-
(2,4-dimethoxyphenyl)acrylate (20). A solution of ethyl 3-(2,4-
dimethoxyphenyl)-3-oxopropanoate (9) (41.91 g, 166 mmol) and tert-
butyl (2-aminoethyl)carbamate (15) (54.7 g, 342 mmol) in ethanol
(180 mL) was treated with acetic acid (16.14 g, 269 mmol) and heated
at reflux. After ∼5 h, the reaction mixture was cooled to ambient
temperature and concentrated under reduced pressure. The resulting
residue was partitioned between ethyl acetate (300 mL) and a 10%
(w/v) aqueous ammonium chloride solution. The organic layer was
separated and washed with water, 10% (w/v) aq ammonium chloride
(3 mL), and brine (10 mL). The organic layer was then agitated with a
saturated aqueous sodium bicarbonate solution, to which brine (6 mL)
was added, and the phases were separated. The organic layer was
washed with brine and dried (Na2SO4). Concentration of the organic
layer afforded 20 as a viscous, amber solid (62.3 g, 95%), which was
used in the subsequent procedure without further purification. 1H
NMR (500 MHz, CDCl3) δ 8.65 (br s, 1 H), 7.12 (d, J = 8.3 Hz, 1 H),
6.50 (dd, J = 8.4, 1.8 Hz, 1 H), 6.47 (d, J = 1.7 Hz, 1 H), 4.88 (br s, 1
H), 4.51 (s, 1 H), 4.14 (q, J = 7.1 Hz, 2 H), 3.83 (s, 6 H), 3.03−3.21
(m, 4 H), 1.43 (s, 9 H), 1.27 (t, J = 7.1 Hz, 3 H). LCMS (ESI+) m/z:
395.4 [M + H]+ (100%).
1
as a colorless solid. H NMR (500 MHz, CD3OD) δ 7.27 (d, J = 8.3
Hz, 1 H), 6.73 (d, J = 2.2 Hz, 1 H), 6.70 (dd, J = 8.3, 2.2 Hz, 1 H),
5.80 (s, 1 H), 4.82 (ddd, J = 14.0, 7.7, 6.4 Hz, 1 H), 4.14 (ddd, J =
14.0, 7.8, 5.9 Hz, 1 H), 3.89 (s, 3 H), 3.87 (s, 3 H), 3.12 (ddd, J = 12.9,
7.7, 6.4 Hz, 1 H), 3.06 (ddd, J = 12.9, 7.8, 5.9 Hz, 1 H). LCMS (ESI+)
m/z: 291.3 [M − NH3 + H]+ (100%), 308.3 [M + H]+ (33%), 615.5
[2M + H]+ (2.3%). HRMS: m/z calcd for C14H18N3O3S [M + H]+
308.1069, found 308.1059. HPLC purity: >95%.
Methyl (Z)-3-(2,4-Dimethoxyphenyl)-3-((2-isopropoxyethyl)-
amino)acrylate (21). A solution of methyl 3-(2,4-dimethoxyphenyl)-
3-oxopropanoate (10) (40.0 g, 168 mmol) and 2-aminoacetamide
hydrochloride (16) (49.4 g, 447 mmol) in methanol (160 mL) was
agitated with the aid of a mechanical stirrer as it was treated with
triethylamine (48 mL, 344 mmol) followed by acetic acid (24 mL, 380
mmol) and then heated in a 80 °C oil bath. After 16 h, the mixture was
allowed to cool to ambient temperature, treated with water (200 mL),
and stirred for 1 h. The resulting solids were collected by filtration and
resuspended in methyl tert-butyl ether (300 mL), filtered, and dried
under a flow of nitrogen gas for 16 h to afford 21 as a flocculent
colorless solid (41.2 g, 83%). 1H NMR (400 MHz, CDCl3) δ 8.77 (t, J
= 5.4 Hz, 1 H), 7.13 (d, J = 8.3 Hz, 1 H), 6.47−6.52 (m, 2H), 4.53 (s,
1 H), 3.84 (s, 3 H), 3.82 (s, 3 H), 3.66 (s, 3H), 3.61 (td, J = 5.5, 5.5
Hz, 2 H), 3.15 (td, J = 5.5, 5.5 Hz, 2 H).
2-(6-(2,4-Dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyr-
imidin-1(2H)-yl)acetamide (5). A suspension of methyl (Z)-3-(2,4-
dimethoxyphenyl)-3-((2-isopropoxyethyl)amino)acrylate (21, 41.2 g,
140 mmol) in 2-methyltetrahydrofuran (200 mL) was treated with
isothiocyanatotrimethylsilane (95 mL, 630 mmol) and then heated at
80 °C for 16 h before the temperature was increased to 100 °C. After
an additional 24 h, the reaction mixture was cooled to ambient
temperature, diluted with methyl tert-butyl ether (300 mL), and the
resulting solid material was collected by vacuum filtration, rinsing with
methyl tert-butyl ether, before drying under a stream of nitrogen gas.
The solid was then dissolved in an aqueous 1 N NaOH solution (600
mL) and extracted twice with dichloromethane. The organic phases
were discarded, and the aqueous layer was cooled in an ice bath before
being acidified with concentrated aqueous HCl. A precipitate began to
form at pH ≈ 7 as the solution was acidified further until pH = 2. The
resulting solid material was collected by vacuum filtration, rinsing with
water, and dried under a stream of nitrogen gas to afford the title
compound 5 as a tan solid (27.8 g, 62%). 1H NMR (500 MHz,
DMSO-d6) δ 12.75 (s, 1 H), 7.31 (br s, 1 H), 7.08 (d, J = 8.54 Hz, 1
H), 6.98 (br s, 1 H), 6.69 (d, J = 2.20 Hz, 1 H), 6.61 (dd, J = 8.54, 2.20
Hz, 1 H), 5.74 (s, 1 H), 5.38 (br s, 1 H), 3.87 (br s, 1 H), 3.82 (s, 3
H), 3.81 (s, 3 H). MS (ES+) m/z: 322.2 [M + H]+. HRMS: m/z calcd
for C14H16N3O4S [M + H]+ 322.0862, found 322.0856. Anal. Calcd for
C14H15N3O4S: C, 52.33; H, 4.71; N, 13.08; S, 9.98. Found: C, 52.09;
H, 4.56; N, 12.86; S, 9.94. HPLC purity: 94.8%.
tert-Butyl 2-(6-(2,4-Dimethoxyphenyl)-4-oxo-2-thioxo-3,4-
dihydropyrimidin-1(2H)-yl)ethylcarbamate (25). A solution of
ethyl (Z)-3-(2-(tert-butoxycarbonylamino)ethylamino)-3-(2,4-
dimethoxyphenyl)acrylate (20) (62.3 g, 158 mmol) in 2-methylte-
trahydrofuran (160 mL) was treated with isothiocyanato-
trimethylsilane (66 mL, 470 mmol) and heated at reflux under
nitrogen. After 15 h, the reaction mixture was cooled to ambient
temperature and quenched by cautious addition of a saturated aqueous
sodium bicarbonate solution (470 mL). The resulting mixture was
extracted with dichloromethane, and the aqueous phase was extracted
twice more with dichloromethane. The combined organic layers were
dried (Na2SO4) and concentrated under reduced pressure to afford a
yellow-amber foam, which was purified by chromatography on silica
gel, eluting with 0−80% ethyl acetate in heptanes to afford 49.2 g of a
solid. This solid was resuspended in equal parts of ethyl acetate and
heptane before being heated at 70 °C for 1 h and then stirred at
ambient temperature for 1 h. The resulting solid was collected by
vacuum filtration, rinsing the material with additional 1:1 ethyl
acetate/heptane, to afford 25 as a colorless fine powder (38.3 g,
Methyl (Z)-3-(2,4-Dimethoxyphenyl)-3-((2-ethoxy-2-
oxoethyl)amino)acrylate (22). A solution of methyl 3-(2,4-
dimethoxyphenyl)-3-oxopropanoate (10) (5.0 g, 21 mmol), glycine
methyl ester hydrochloride (17) (10.5 g, 83.9 mmol), acetic acid (1.20
mL, 21 mmol), and triethylamine (8.5 g, 83.9 mmol) in ethanol (30
mL) was heated at 100 °C for 18 h. The reaction mixture was allowed
to cool to ambient temperature and partitioned between EtOAc and
saturated aqueous ammonium chloride. The organic layer was
1
59.5%). H NMR (500 MHz, CDCl3, major rotamer) δ 9.58 (br s, 1
H), 7.26 (d, J = 8.4 Hz, 1 H), 6.59 (dd, J = 8.4, 2.1 Hz, 1 H), 6.51 (d, J
K
DOI: 10.1021/acs.jmedchem.5b00963
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Journal of Medicinal Chemistry
Article
separated, washed with brine, dried over sodium sulfate, and
concentrated under reduced pressure. The residue was dissolved in
dichloromethane (10 mL) and eluted through a column of silica gel
with a gradient of ethyl acetate in heptanes (15−35%) to afford 22 as a
yellow solid (4.7 g, 69%). The material was used directly in the next
step without further purification. 1H NMR (400 MHz, CDCl3) δ 8.95
(br s, 1 H), 7.14 (d, J = 10.57 Hz, 1 H), 6.49 (dd, J = 8.28, 2.07 Hz, 1
H), 6.46 (d, J = 2.07, 1 H), 4.60 (s, 1 H), 4.16 (q, J = 7.80 Hz, 2 H),
3.83 (s, 3 H), 3.80 (s, 3 H), 3.69 (s, 3 H), 1.24 (t, J = 7.80 Hz, 3 H).
MS (ES+) m/z: 324.3 [M + 1]+.
solid was suspended in ethyl acetate (350 mL), heated to 70 °C under
a stream of nitrogen gas with stirring for 1 h and then at ambient
temperature for 18 h before the suspension was cooled down to 0 °C,
and the solid was collected by vacuum filtration, the filter cake washed
with cold (0 °C) ethyl acetate (50 mL) and dried in the vacuum oven
at 50 °C for 9 h to give 28 (45.4 g, 78.8%) as an off-white powder. 1H
NMR (500 MHz, CD3OD) δ 8.99 (br s, 1 H), 7.16 (d, J = 7.65 Hz, 1
H), 6.65 (s, 1 H), 6.62 (d, J = 7.65 Hz, 1 H), 5.78 (s, 1 H), 5.51−5.41
(m, 1 H), 4.22−4.14 (m, 1 H), 3.87 (s, 3 H), 3.85 (s, 3 H), 3.19−3.11
(m, 2 H), 3.06−3.00 (m, 2 H), 1.42 (s, 9 H). MS (ES+) m/z: 465.3 [M
+ H]+.
N-(2-Aminoethyl)-2-(6-(2,4-dimethoxyphenyl)-4-oxo-2-thio-
xo-3,4-dihydropyrimidin-1(2H)-yl)acetamide Hydrochloride
(6). Ethanol (21.5 mL), cooled at 0 °C under nitrogen, was slowly
treated with acetyl chloride (1.55 mL) over 5 min, after which the
reaction mixture was heated at 50 °C. After 30 min, the reaction
mixture was cooled to ambient temperature and tert-butyl-(2-(2-(6-
(2,4-dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-
yl)acetamido)ethyl)carbamate (28) (1.0 g, 2.15 mmol) was added,
followed by heating to 50 °C. After 1 h, the reaction mixture was
cooled to ambient temperature and concentrated under reduced
pressure. The resulting residue was then suspended in ethanol (10
mL), heated at 75 °C for 20 min before adding ethyl acetate (20 mL).
After heating for another 20 min, the reaction mixture was allowed to
gradually cool to ambient temperature with stirring. After 18 h, the
resulting precipitate was filtered and dried in a vacuum oven at 70 °C
for 20 h to afford 6 (751 mg, 87%) as a colorless solid. 1H NMR (500
MHz, DMSO-d6) δ 12.81 (br s, 1 H), 8.26 (br s, 1 H), 8.01 (br s, 2
H), 7.08 (d, J = 7.91 Hz, 1 H), 6.70 (s, 1 H), 6.62 (d, J = 7.91 Hz, 1
H), 5.78 (s, 1 H), 5.41−5.35 (m, 1 H), 4.07−4.02 (m, 1 H), 3.84 (s, 3
H), 3.83 (s, 3 H), 3.20−3.16 (m, 2 H), 2.74−2.64 (m, 2 H). MS (ES+)
m/z: 365.2 [M + H]+. HRMS: m/z calcd for C16H21N4O4S [M + H]+
365.1284, found 365.1278. HPLC purity: >95%.
Sodium 1-(2,5-Dimethoxyphenyl)-3-ethoxy-3-oxoprop-1-
en-1-olate (11). A 20 L reaction vessel was charged with magnesium
ethoxide (413.5 g, 3.61 mol) and THF (6.6 L), and the resulting
mixture was treated with ethyl hydrogen malonate (29) (888.9 mL,
7.23 mol) in THF (20 mL) and heated at 45 °C for 4 h. Meanwhile, a
20 L reactor was charged with 2,5-dimethoxybenzoic acid (30) (600 g,
3.29 mol) and THF (3.6 L). To this mixture was added 1,1′-
carbonyldiimidazole (585.98 g, 3.61 mol) in portions to avoid excess
foaming. After stirring for 3 h at ambient temperature, the solution of
activated acid was added gradually to the ethyl malonate solution and
the combined reaction mixture was heated at 45 °C. After 20 h, the
mixture was concentrated under reduced pressure followed by the
addition of ethyl acetate (6 L) and 2 N HCl (3 L). After mixing, the
layers were separated and the organic phase was washed sequentially
with 2 N HCl (3 L), saturated sodium bicarbonate (3 L), and water (3
L). The organic phase was concentrated under reduced pressure, the
residue taken up in ethyl acetate (6 L) and concentrated again to
afford an oil, which was transferred to a 20 L reaction vessel with 5 L
of ethyl acetate and treated with a 4.35 M solution of sodium
methoxide (793 mL, 3.45 mol) in methanol. After stirring at room
temperature for 3 h, an additional 6 L of ethyl acetate was added, the
solid was collected by vacuum filtration and dried overnight in a
vacuum oven at 40 °C to give 661 g of 11 (73%) as a solid. 1H NMR
(400 MHz, DMSO-d6) δ 6.92 (d, J = 3.0 Hz, 1 H), 6.84 (d, J = 8.8 Hz,
1 H), 6.73 (dd, J = 8.8, 3.0 Hz, 1 H), 4.67 (s, 1 H), 3.88 (q, J = 7.0 Hz,
2 H), 3.67 (s, 6 H), 1.12 (t, J = 7.0 Hz, 3 H). MS (ES+) m/z: 253.1 [M
+ H]+.
Ethyl 2-(6-(2,4-Dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)acetate (26). A solution of methyl (Z)-3-
(2,4-dimethoxyphenyl)-3-((2-ethoxy-2-oxoethyl)amino)acrylate (22)
(4.68 g, 15.1 mmol) in 2-methyltetrahydrofuran (38 mL) was treated
with isothiocyanatotrimethylsilane (12.9 mL, 90.8 mmol), and the
solution was purged with nitrogen gas and heated at 110 °C. After 18
h, the mixture was allowed to cool to ambient temperature and
concentrated under reduced pressure to afford a red solid, which was
then suspended in 200 mL of a 25% ethyl acetate in heptanes mixture.
After stirring at room temperature for 1 h, the solid was collected by
filtration and then triturated with methylene chloride (100 mL),
concentrated under reduced pressure, and dried under vacuum to
afford 26 (4.42 g, 87%) as a pink solid. This material was used directly
1
in the next step without further purification. H NMR (500 MHz,
CDCl3) δ 9.91 (br s, 1 H), 7.13 (d, J = 6.12 Hz, 1 H), 6.54 (s, 1 H),
6.51 (d, J = 6.12 Hz, 1 H), 5.86 (s, 1 H), 5.44−5.40 (m, 1 H), 4.25−
4.20 (m, 1 H), 4.16−4.06 (m, 2 H), 3.86 (s, 3 H), 3.83 (s, 3 H), 1.20
(t, J = 6.12 Hz, 3 H). MS (ES+) m/z: 351.5 [M + H]+.
2-(6-(2,4-Dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyr-
imidin-1(2H)-yl)acetic Acid (27). A solution of ethyl 2-(6-(2,4-
dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)-
acetate (26) (6.8 g, 20.3 mmol) in methanol (34 mL) was treated with
a 6 N aqueous NaOH solution (16.9 mL), and the solution was heated
at 35 °C. After 3 h, the reaction mixture was allowed to cool to
ambient temperature and concentrated under reduced pressure. The
residue was combined with water (100 mL) and extracted with ethyl
acetate (2 × 200 mL), and the aqueous phase was acidified to pH ≈ 2
with concentrated aqueous HCl. The acidic aqueous solution was
extracted with ethyl acetate (3 × 200 mL), and the combined organic
phases were dried (Na2SO4) and concentrated under reduced pressure
to afford 6.53 g (99%) of 27 as a colorless solid. 1H NMR (500 MHz,
CD3OD) δ 7.16 (d, J = 8.86 Hz, 1 H), 6.67 (s, 1 H), 6.64 (d, J = 8.86
Hz, 1 H), 5.79 (s, 1 H), 5.52−5.40 (m, 1 H), 4.34−4.19 (m, 1 H), 3.87
(s, 3 H), 3.86 (s, 3 H). MS (ES+) m/z: 323.2 [M + H]+.
tert-Butyl (2-(2-(6-(2,4-Dimethoxyphenyl)-4-oxo-2-thioxo-
3,4-dihydropyrimidin-1(2H)-yl)acetamido)ethyl)carbamate
(28). A solution of 2-(6-(2,4-dimethoxyphenyl)-4-oxo-2-thioxo-3,4-
dihydropyrimidin-1(2H)-yl)acetic acid (27) (40 g, 124 mmol) in
DMF (300 mL) was treated with tert-butyl-(2-aminoethyl)carbamate
(40 g, 250 mmol) and pyridine (30 mL) and cooled to 0 °C under
nitrogen. A 50% solution of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphos-
phorinane 2,4,6-trioxide in DMF (109 mL) was carefully added with
stirring. After 1 h, the cooling bath was removed and the reaction
mixture was stirred at ambient temperature. After 4 h, the solution was
poured slowly into a stirring 0.5 M aqueous HCl solution (2500 mL).
After stirring at ambient temperature for 1 h, the resulting solid was
collected by vacuum filtration. The solid was washed with 0.5 M
aqueous HCl (500 mL) followed by water (500 mL). The resulting
solid was dried in a vacuum oven at 50 °C for 20 h to afford 54.6 g of
light beige powder. This solid was suspended in ethyl acetate (500
mL), heated to 70 °C under a stream of nitrogen gas with stirring for 1
h, and then allowed to cool to ambient temperature with stirring. After
18 h, the suspension was cooled to 0 °C, the solid was collected by
vacuum filtration, the filter cake washed with cold (0 °C) ethyl acetate
(100 mL) and dried in the vacuum oven at 50 °C for 9 h to afford 49.0
g of an off-white solid. This solid was suspended in acetonitrile (300
mL) and stirred at 70 °C under a stream of nitrogen for 18 h. The
mixture was cooled to 0 °C, and the resultant solid was collected by
vacuum filtration, washed with cold acetonitrile (50 mL), and dried in
a vacuum oven at 50 °C for 8 h to give 46.5 g of off-white solid. This
Ethyl (Z)-3-((2-Amino-2-oxoethyl)amino)-3-(2,5-
dimethoxyphenyl)acrylate (23). A 5 L reaction vessel was charged
with methanol (3.3 L), sodium methoxide (102.4 g, 1.8 mol), and
aminoacetamide hydrochloride (16) (202 g, 1.8 mol). The mixture
was heated at 65 °C for 1 h before cooling to 50 °C and adding acetic
acid (30.88 g, 29.47 mL, 514.25 mmol) and sodium 1-(2,5-
dimethoxyphenyl)-3-ethoxy-3-oxoprop-1-en-1-olate (11) (300 g, 1.09
mol). After heating at reflux for 16 h, the reaction mixture was cooled
to 10 °C, stirred for 30 min, the resulting solid collected by filtration
and dried in a vacuum oven for 14 h to afford 23 (339.4 g, 100%) as a
L
DOI: 10.1021/acs.jmedchem.5b00963
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
solid. 1H NMR (400 MHz, DMSO-d6) δ 8.84 (t, J = 4.7 Hz, 1 H), 7.36
(s, 1 H), 7.09 (s, 1 H), 7.02 (d, J = 8.9 Hz, 1 H), 6.97 (dd, J = 8.9, 2.8
Hz, 1 H), 6.74 (d, J = 2.8 Hz, 1 H), 4.31 (s, 1 H) 4.03 (q, J = 7.1 Hz, 2
H), 3.74 (s, 6 H), 3.58 (br s, 1 H), 3.47 (br s, 1 H), 1.18 (t, J = 7.1 Hz,
3 H). MS (ES+) m/z: 309.1 [M + H]+.
2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)acetamide (8). A reaction vessel equipped
with an efficient stirrer was charged with ethyl (Z)-3-((2-amino-2-
oxoethyl)amino)-3-(5-chloro-2-methoxyphenyl)acrylate (24) (15 g,
50.2 mmol), butyl acetate (150 mL), and isothiocyanatotrimethylsilane
(21.1 g, 22.7 mL, 160.7 mmol), and the mixture was heated to reflux.
After 15 h, the mixture was cooled to 30 °C and treated with 1 N
aqueous NaOH (112.5 mL). After 30 min, the organic layer was
separated and extracted with another portion of 1 N sodium hydroxide
(37.5 mL). The combined aqueous phases were extracted twice with
dichloromethane (2 × 45 mL), filtered, and treated with 6 N HCl until
a pH of 2.5 was achieved. After stirring for 1 h, the resulting solid was
collected by vacuum filtration, resuspended in 100 mL of a 1:1
methanol−water solution, heated with stirring at 50 °C for 2 h, and
cooled to room temperature before collecting the solid by filtration
and drying in a vacuum oven (20 mmHg, 50 °C) for 12 h to afford 8
2-(6-(2,5-Dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyr-
imidin-1(2H)-yl)acetamide (7). A 5 L reaction vessel equipped with
an efficient stirrer was charged with ethyl (Z)-3-((2-amino-2-
oxoethyl)amino)-3-(2,5-dimethoxyphenyl)acrylate (23) (400 g, 1.30
mol), butyl acetate (3.4 L), and isothiocyanatotrimethylsilane (585.67
mL, 544.96 g, 4.15 mol), and the mixture was heated to reflux. After 16
h, the mixture was cooled to 40 °C and treated with a solution of 2 N
aqueous NaOH (1.95 L). The organic layer was separated and
extracted with another portion of 2 N aqueous NaOH (0.32 L). The
combined aqueous phases were filtered, extracted twice with
dichloromethane (2 × 1.6 L), and added slowly to a well-stirred 3
N aqueous HCl solution (1.3 L) at room temperature. After stirring
for 30 min, the resulting solid was filtered and dissolved in
dimethylformamide (2.4 L) at 90 °C and before water (2 L) was
added slowly to the solution. The mixture was cooled gradually to
room temperature and the resulting solid isolated by vacuum filtration,
rinsing with water. This solid was then suspended in 1.25 L of
methanol and stirred as 1.25 L of water was added. The mixture was
heated with stirring at 50 °C for 2 h and then cooled to 10 °C for 2 h
before collecting the solid by vacuum filtration and drying in a vacuum
oven to afford 7 (245 g, 59%) as a solid. 1H NMR (500 MHz, DMSO-
d6) δ 12.80 (s, 1 H), 7.32 (br s, 1 H), 7.06−7.11 (m, 2 H), 7.06 (br s, 1
H), 6.74−6.77 (m, 1 H), 5.82 (d, J = 2.20 Hz, 1 H), 5.37 (br s, 1 H),
3.88 (br s, 1 H), 3.78 (s, 3 H), 3.70 (s, 3 H). MS (ES+) m/z: 322.2 [M
+ H]+. HRMS: m/z calcd for C14H16N3O4S [M + H]+ 322.0862, found
322.0856. HPLC purity: >95%.
1
(8.7 g, 53%) as a tan solid (mp = 165.3 °C). H NMR (500 MHz,
DMSO-d6) δ 12.85 (s, 1 H), 7.57 (dd, J = 9.03, 2.68 Hz, 1 H), 7.33 (s,
1 H), 7.17−7.23 (m, 2 H), 7.10 (s, 1 H), 5.89 (d, J = 1.71 Hz, 1 H),
5.41 (br s, 1 H), 3.89 (br s, 1 H), 3.84 (s, 3 H). MS (ES+) m/z: 326.0
[M + H]+. HRMS: m/z calcd for C13H13ClN3O3S [M + H]+ 326.0366,
found 326.0361. Anal. Calcd for C13H12ClN3O3S: C, 47.93; H, 3.71;
N, 12.90; S, 9.84. Found: C, 47.81; H, 3.70; N, 12.83; S, 9.83. HPLC
purity: >95%.
Pharmacokinetics. All experiments involving animals were
conducted in our AAALAC-accredited facilities and were reviewed
and approved by Pfizer Institutional Animal Care and Use Committee.
Male CD-1 mice (25−35 g), jugular vein/carotid artery double
cannulated Wistar-Han rats (250 g), obtained from Charles River
Laboratories (Wilmington, MA), male beagle dogs (12 kg), and male
cynomolgus monkeys (7 kg) were used for these studies. Animals were
fasted overnight and fed after dosing (1.0 or 2.0 h), whereas access to
water was provided ad libitum. Compounds were administered
intravenously (iv) in either saline, 12% sulfobutyl ether β-cyclodextrin
with or without 1% DMSO or 30% hydroxypropyl-β-cyclodextrin
containing 5% DMSO via the tail vein (mice, n = 3), carotid artery
(rats, n = 3), saphenous vein (dogs, n = 3), femoral vein (monkeys, n =
2) at a dose of 1.0 mg/kg in a dosing volume of 5 mL/kg (mice), 2
mL/kg (rats), 0.5 mL/kg (dogs), and 1 mL/kg (monkeys). Serial
blood samples were collected before dosing and 0.083, 0.25, 0.5, 1, 2,
4, 7, and 24 h after dosing. Compounds were also administered by po
gavage to mice (5 mg/kg at 5 mL/kg in 0.5% methylcellulose), rats (5
mg/kg at 5 mL/kg in 0.5% methylcellulose), dogs (3 mg/kg at 1.5
mL/kg in 0.5% methylcellulose), and monkeys (3 mg/kg at 5 mL/kg
in 0.5% methylcellulose). Blood samples were taken prior to po
administration, and then serial samples were collected at 0.25, 0.5, 1, 2,
4, 7, and 24 h after dosing. Blood samples from the various
pharmacokinetic studies were centrifuged to generate plasma. All
plasma samples were kept frozen until analysis. Urine samples (0−7.0
and 7.0−24 h; pooled 0−24 h for mice) were also collected after iv
administration to mice, rats, dogs, and monkeys. For mouse samples,
aliquots of plasma (10 μL) or urine (2 μL mixed with 8 μL plasma)
were extracted using liquid/liquid extraction with methyl tert-butyl
ether containing internal standard. For rat, dog, or monkey samples,
aliquots of plasma or urine (20−25 μL) were transferred to 96-well
blocks and acetonitrile (150−200 μL) containing internal standard was
added to each well. Supernatant was dried under nitrogen and
reconstituted with 100 μL of acetonitrile/water (1:1 with 0.1% formic
acid for mice; 20:80 for dog) or added to 100 μL of water without
evaporation (rat, monkey). Following extraction, the samples were
then analyzed by LC−MS/MS, and concentrations of analyte in
plasma and urine were determined by interpolation from a standard
curve as described previously.39
Ethyl 3-(5-Chloro-2-methoxyphenyl)-3-oxopropanoate (12).
A 3 L three-necked round-bottomed flask, flushed with nitrogen, was
charged with magnesium ethoxide (67.46 g, 589.51 mmol) and THF
(1100 mL), and the resulting mixture was stirred as ethyl hydrogen
malonate (29) (162.26 g, 1.18 mol) in THF (100 mL) was added and
the mixture was heated at 45 °C for 4 h. Meanwhile, a 2 L three-
necked round-bottomed flask, flushed with nitrogen, was charged with
5-chloro-2-methoxybenzoic acid (31) (100 g, 536 mmol) and THF
(600 mL). To this mixture was added 1,1′-carbonyldiimidazole (95.59
g, 589.5 mmol) in portions to avoid excess foaming. After stirring for 3
h, the solution of activated acid was added gradually to the ethyl
malonate solution, and the resulting reaction mixture was heated at 45
°C. After 20 h, the reaction mixture was concentrated under reduced
pressure before adding ethyl acetate (1 L) followed by 2 N HCl (500
mL). After mixing, the layers were separated and the organic phase was
washed sequentially with 2 N HCl (500 mL), saturated sodium
bicarbonate (500 mL), and water (500 mL). The organic phase was
concentrated under reduced pressure to afford 12 (104.94 g, 76%) as a
solid. 1H NMR showed the desired product as a 7.5:1 keto/enol
1
mixture. For the keto tautomer: H NMR (500 MHz, CDCl3) δ 7.85
(d, J = 2.93 Hz, 1 H), 7.45 (dd, J = 8.90, 2.81 Hz, 1 H), 6.92 (d, J =
8.78 Hz, 1 H), 4.18 (q, J = 7.16 Hz, 2 H), 3.95 (s, 2 H), 3.90 (s, 3 H),
1.24 (t, J = 7.07 Hz, 3 H). MS (ES+) m/z: 257.2 [M + H]+.
Ethyl (Z)-3-((2-Amino-2-oxoethyl)amino)-3-(5-chloro-2-
methoxyphenyl)acrylate (24). A 5 L reaction vessel was charged
with methanol (3.3 L), sodium methoxide (102.4 g, 1.8 mol), and
aminoacetamide hydrochloride (16) (202 g, 1.8 mol). The mixture
was heated at 65 °C for 1 h before cooling to 50 °C and adding acetic
acid (514.3 mmol, 30.9 g, 29.5 mL) and ethyl 3-(5-chloro-2-
methoxyphenyl)-3-oxopropanoate (12) (300 g, 1.17 mol). After
heating at reflux for 16 h, the reaction mixture was stirred as it was
cooled to 10 °C. After 30 min the resulting solid was filtered and dried
in a vacuum oven (20 mmHg, 65 °C) for 14 h to afford 24 (339.4 g,
In Vitro Metabolism of N1-Substituted-6-arylthiouracils in
MPO. Stock solutions of thiouracil derivatives were prepared in
DMSO. Compounds (final concentration of 10 μM) were added to an
incubation mixture containing 50 mM sodium phosphate buffer, pH
7.4, 150 mM NaCl, 1 mM diethylenetriaminepentacetic acid, 2 μM
H2O2, 30 μM Amplex Red, and 1 mM GSH. Reactions were initiated
by the addition of 100 pM MPO, and the final concentration of
1
93%) as a solid. H NMR (500 MHz, DMSO-d6) δ 8.80 (t, J = 5.00
Hz, 1 H), 7.47 (dd, J = 8.90, 2.81 Hz, 1 H), 7.27 (br s, 1 H), 7.22 (d, J
= 2.68 Hz, 1 H), 7.14 (d, J = 8.78 Hz, 1 H), 7.09 (br s, 1 H), 4.30 (s, 1
H), 4.03 (q, J = 7.07 Hz, 2 H), 3.80 (s, 3 H), 3.56 (br s, 1 H), 3.45 (br
s, 1 H), 1.18 (t, J = 7.07 Hz, 3 H). MS (ES+) m/z: 313.2 [M + H]+.
M
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DMSO was kept at 2% in the incubation (volume of 200 μL). The
reaction mixture was incubated at 37 °C for 60 min, terminated by the
addition of ice cold acetonitrile (400 μL) and centrifuged (3000g, 15
min). The supernatants were dried under a steady stream of nitrogen,
reconstituted with 25% aqueous acetonitrile (250 μL), and analyzed
via LC−MS/MS for formation of sulfydryl conjugates. Qualitative
assessment of metabolites was conducted using a Thermo Finnegan
Surveyor photodiode array plus detector, Thermo Acela pump, and a
Thermo Acela autosampler (Thermoscientific, West Palm Beach, FL).
Chromatography was performed on a Phenomenex Hydro RP C18
(4.6 mm × 150 mm, 3.5 μm) column (Phenomenex, Torrance, CA).
The mobile phase was composed of 5 mM ammonium formate buffer
with 0.1% formic acid (pH = 3) (solvent A) and acetonitrile (solvent
B) at a flow rate of 1 mL/min. The binary gradient was as follows:
solvent A to solvent B ratio was held at 95:5 (v/v) for 3 min and then
adjusted to 55:45 (v/v) from 0 to 35 min, 30:70 (v/v) from 35 to 45
min, and 5:95 (v/v) from 45 to 52 min where it was held for 3 min
and then returned to 95:5 (v/v) for 6 min before next analytical run.
Metabolite identification was performed on a Thermo Orbitrap mass
spectrometer operating in positive ion electrospray mode. The spray
potential was 4 V, and heated capillary was at 275 °C. Xcalibur
software version 2.0 was used to control the HPLC−MS system. Full
scan measurements from 50−1000 amu were collected at 15 000
resolution, with two data-dependent acquisition scans of the most
intense ion in scan events 1 and 2, respectively. Product ion spectra
were acquired at a normalized collision energy of 65 eV with an
isolation width of 2 amu.
Liquid chromatography−tandem mass spectrometry (LC−MS/MS)
analysis of an incubation of arylthiouracil 2 (retention time (tR) = 25.4
min, exact mass (MH+) = 351.1367) with human MPO/H2O2/Cl− in
the presence of excess glutathione (GSH) (1 mM) indicated the
formation of a GSH conjugate (tR = 15.7 min; MH+ = 624.2334, m/z
(+H+) = 495.1906 (loss of pyroglutamate, 129 amu), which can be
obtained via oxidation of the thiocarbonyl group in 2 to the putative
sulfonate species followed by nucleophilic displacement in the
presence of exogenously added GSH (Scheme 2).
Inhibition of MPO Activity in LPS-Treated Cynomolgus
Monkeys by 8. All experiments involving animals were conducted in
our AAALAC-accredited facilities and were reviewed and approved by
Pfizer Institutional Animal Care and Use Committee. Male
cynomolgus monkeys (n = 8; >2.5 years old at initiation of dosing)
were assigned to dose groups randomized in a crossover manner with
2 weeks between treatments. The treatments were the following: iv
LPS (5 μg/kg; Sigma, L3491) in sterile saline followed after 1 h by po
administration of vehicle (20 mM Tris, 0.5% [w/v] hydroxypropyl-
methylcellulose, and 0.5% [w/v] hydroxypropylmethylcellulose acetate
succinate high-fine grade) or 8 in vehicle at 5, 20, or 80 mg/kg. Blood
samples were taken just prior to LPS dosing and at 0.5, 2.0, 4.0, 8.0,
and 24 h after vehicle or 8 dosing in sodium heparinized containers.
Total MPO and residual MPO activity were determined from plasma
samples by antibody capture as described above for the human whole
blood assay, and plasma levels of 8 were measured by liquid
chromatography−tandem mass spectrometry.
ABBREVIATIONS USED
■
MPO, myeloperoxidase; H2O2, hydrogen peroxide; COPD,
chronic obstructive pulmonary disease; TPO, thyroid perox-
idase; CYP, cytochrome P450; PF-06282999, 2-(6-(5-chloro-2-
methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-
yl)acetamide; po, oral; THF, tetrahydrofuran; LPS, lip-
opolysaccharide; PTU, propylthiouracil; kinact, maximal rate of
MPO inactivation; KI, concentration required to achieve half
the maximal rate of inactivator; [I], inhibitor concentration;
TMSNCS, isothiocyanatotrimethylsilane; T3P, 2,4,6-tripropyl-
1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide; LC−MS/MS,
liquid chromatography−tandem mass spectrometry; GSH,
glutathione; ADME, absorption, distribution, metabolism, and
excretion; HLM, human liver microsome; HHEP, cryopre-
served human hepatocyte; RRCK, Ralph−Russ canine kidney;
iv, intravenous; CLp, plasma clearance; Vdss, steady state volume
of distribution; t1/2, elimination half-life; Tmax, time to reach
Cmax
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Corresponding Author
*Phone: 860-441-3535. E-mail: Roger.B.Ruggeri@pfizer.com.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare the following competing financial
interest(s): All authors are current or former employees of
Pfizer, Inc.
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